Three-dimensional Structures Of Biological Macromolecules

生物大分子的三维结构

• 批准号: 7594372
• 负责人: Bernard R Brooks
• 金额: $ 29.92万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594372
• 关键词: Acetyl Coenzyme A; Active Sites; Adult; Algorithms; Architecture; Binding; Biological; Blood Transfusion; C-terminal; Cell membrane; Cells; Chemicals; Chemoreceptors; Chemotaxis; Citric Acid Cycle; Class; Classification; Code; Communities; Complex; Computational Technique; Computer software; Computing Methodologies; Cryoelectron Microscopy; Crystallography; Cysteine; DNA; Data; Development; Disease; Docking; Electron Microscopy; Electrons; Embryonic Development; Enzymes; Escherichia coli; Genes; Glycolysis; Glycoproteins; Goals; HIV; HIV Envelope Protein gp120; HIV-1; Hematological Disease; Histocompatibility Antigens Class II; Homeodomain Proteins; Human; Image; Immune response; Individual; Integrins; Laboratories; Ligands; Link; Maps; Membrane; Metabolism; Methods; Microscopic; Modeling; Molecular; Molecular Conformation; Molecular Structure; Monte Carlo Method; Multienzyme Complexes; NMR Spectroscopy; Noise; Organ; Organogenesis; Output; Pattern; Peptides; Peroxides; Phosphorylation; Phosphotransferases; Play; Pregnancy; Prostate; Protein Region; Proteins; Pyruvate; Pyruvate Dehydrogenase Complex; Pyruvates; Range; Reactive Oxygen Species; Reporting; Research; Resolution; Roentgen Rays; Role; SIV; SLC26A3 gene; Sampling; Signal Transduction; Spectrum Analysis; Stem cells; Structure; Support of Research; Surface; System; Techniques; Technology; Temperature; Therapeutic; Thinking; Time; Tomogram; Transplanted tissue; Vaccines; Variant; Viral; Virion; Weight; Work; X ray spectroscopy; X-Ray Crystallography; base; carcinogenesis; density; design; dimer; disease-causing mutation; electron density; electron tomography; homeodomain; human EML2 protein; image processing; inorganic phosphate; macromolecular assembly; macromolecule; molecular assembly/self assembly; molecular modeling; molecular size; neonate; particle; periplasm; peroxiredoxin; prophylactic; protein degradation; protein structure; pyruvate dehydrogenase; receptor; research study; simulation; structural biology; three dimensional structure; three-dimensional modeling; tomography; tool; transcription factor; tumorigenesis

1. Using molecular modeling, CD and NMR spectroscopy. The three-dimensional structures of NK-2 class homeodomain proteins, the human NKX 2-1, NKX 2-5, and NKX 3-1 proteins, have been investigated by Dr. Gruschus in the free state, bound to DNA, and in ternary complexes with associated transcription factors. These proteins orchestrate organogenesis during embryonic development, and maintain organ cells in their differentiated state in adults, suppressing carcinogenesis. Recently in our laboratories, stabilizing and destabilizing interactions have been discovered between the homeodomain and motifs in the N and C-terminal flexible random coil regions. These motif interactions appear to be modulated by phosphorylation. The stuctural mechanisms by which these interactions stabilize and destabilize the homeodomain are our current focus. Additional results tie homeodomain stability with protein turnover in the cell. Thus, these N and C-terminal motifs and their interacting kinases could be targets for therapies aimed at modulating homeodomain protein levels. In particular, increasing NKX 3-1 stability is an important therapeutic goal since lowered NKX 3-1 protein levels have been implicated in prostate tumorigenesis. 2. Electron Microscopy and Tomography Image Processing. The EMAP module in CHARMM has been developed by Dr. Wu and is becoming a very useful tool in electron microscopy structure determination. This module employs the Grid-Threading Monte Carlo method to search for the best fitting structure and is highly efficient and reduces the fitting time by one or two orders of magnitude as compared with other software available in this field. 2.1. Structure study through single-particle cryoelectron microscopy. The pyruvate dehydrogenase (PDH)2 multienzyme complexes play a central role in cellular metabolism,catalyzing the oxidative decarboxy-lation of pyruvate to acetyl CoA,to link glycolysis and the tricarboxylic acid cycle. Here,we extend our previous studies with the report of the architecture of a 1:1 stoichiometric complex containing 60 copies each of the E2 and E3 enzymes.We present a cryoelectron microscopic analysis of the reconstructed three-dimensional structure of the purified E2E3 complex and use automated docking methods to interpret the density map in terms of the probable localization of the E2 and E3 molecules. Our findings show that the arrangement of E1 and E3 molecules in the outer shell of the pyruvate dehydrogenase complex are remarkably similar and indicate that the design of the annular gap allows the lipoyl domain to have access to the active sites of E1,E2,and E3 enzymes from within the annular gap. 2.2 Structure determination through single-particle electron tomography Single-particle electron tomography can be used to obtain 3D volume data of molecular systems without averaging many particles. This technology has the potential to study multiple conformational states of single particles. High noise due to limited sampling and distorsion caused by missing widges are two difficulties for this technology. Normal fitting methods often fail when dealing with such a high noisy data. The core-weighted fitting method we developed and implemented into CHARMM can tolerate such high noise and produce reasonable fitting results. We are applying the core-weighted fitting method to derive complex structures from single-particle electron tomography data. PDH complexes based on icosahedral symmetry are among the largest cellular machines and are a good candidate for single-particle electron tomography study. With the tomography image, we successfully docked the E2CD core and E1 domains and obtained complexes that agree with the results from cro-EM studies. These results validate the experiment techniques as well as our fitting methods for tomography data. Another ongoing project is to determine the structure of the Env spikes of HIV. The Env spikes of HIV-1 are composed of surface envelope glycoprotein (gp120SU) and transmembrane envelope glycoprotein (gp41TM) subunits, which, in their native configuration, elicit partially effective humoral immune responses and represent obvious vaccine targets for viral prophylactics. The Env spikes are thought to be trimeric and structure-based models have been proposed. However, despite intensive efforts, the arrangement and orientation of the loop-deleted gp120 core atomic structures within a native Env spike and their association with the gp41 subunits have remained largely speculative. Recently, Dr. Subramaniams lab obtained electron tomograms of the Env spikes on intact, chemically fixed SIV and HIV-1. We are now applying our image processing and fitting methods to reveal the distribution pattern of Env spikes on individual virions and to generate a three-dimensional (3D) model of the SIV Env spike. 2.3 Structure determination through of Chemotaxis receptor assemblies The image processing methods developed in our lab has been successfully applied in determination of the three-dimensional structure of a chemotoaxis receptor in E-Coli cell membrane by use of electron tomography and averaging of segmented volume densities from electron tomogram. This work bridges the gap between crystallography and cryoelectron tomography of whole cells and makes a major advance in understanding of bacterial chemotaxis. Although atomic structures of both periplasmic and crytoplasmic parts of bacterial chemoreceptors have been determined, the integral structure of the entire membrane-embedded molecules remains unresolved, as does receptor arrangement in the higher-order oligomeric complexes. Our results confirm previously suggested trimer-of-dimers arrangement of receptors. In addition, we reveals two conformational states of this signal transduction machinery, providing direct structural information in the study of signal transduction mechanism. 3. Protein-protein docking with Map Objects. We developed a method that uses map objects for molecular modeling to efficiently derive structural information from experimental maps, as well as conveniently manipulate map objects, perform conformational search directly using map objects. This development work has been implemented into CHARMM. This implementation enables CHARMM to manipulate map objects, including map input, output, comparison, docking, etc. Particularly, we implemented the core-weighted correlation functions to effectively recognize correct fit of component maps in complex maps, and the grid-threading Monte Carlo search algorithm to efficiently construct complex structures from electron density maps. Dr. James M . Gruschus is applying this method in his structure study of the peroxiredoxin complex. Peroxiredoxins (Prx) are one of several classes of proteins that reduce peroxides, reactive oxygen species produced as a by-product of cellular metabolism. For modeling the complex, the ATP-bound human Sulfiredoxin (hSrx) was docked to hyperoxidized Prx II using EMAP. In the docked structures with the Prx cysteine-sulfinic residue closest to the hSrx reactive cysteine, Asn186 of Prx II is in contact with the hSrx-bound ATP beta and gamma phosphate groups. 4. Xray Crystallography Dr. Parry has reported the structure of the class II major histocompatibility complex DRA, DRB3 in complex with an integrin peptide (PDB code 2Q6W). This is the first structure from the DRB3 gene locus. The structure models at high resolution the molecular basis of severe blood diseases associated with pregnancy, neonates, blood transfusion, stem cell and tissue transplant. A second structure is a complex with an altered ligand has been refined and awaits submission.

1. 使用分子建模、CD 和 NMR 光谱。 Gruschus 博士研究了 NK-2 类同源域蛋白（人类 NKX 2-1、NKX 2-5 和 NKX 3-1 蛋白）的游离状态、与 DNA 结合的状态以及在与相关转录因子的三元复合物。这些蛋白质在胚胎发育过程中协调器官发生，并在成人中维持器官细胞处于分化状态，从而抑制癌变。最近在我们的实验室中，在 N 和 C 末端柔性随机卷曲区域的同源结构域和基序之间发现了稳定和不稳定的相互作用。这些基序相互作用似乎受到磷酸化的调节。这些相互作用稳定和破坏同源结构域的结构机制是我们当前的重点。其他结果将同源结构域稳定性与细胞中蛋白质周转联系起来。因此，这些 N 和 C 末端基序及其相互作用的激酶可以成为旨在调节同源域蛋白水平的治疗的靶标。 特别是，增加 NKX 3-1 稳定性是一个重要的治疗目标，因为降低的 NKX 3-1 蛋白水平与前列腺肿瘤发生有关。 2. 电子显微镜和断层扫描图像处理。 CHARMM 中的 EMAP 模块由吴博士开发，正在成为电子显微镜结构测定中非常有用的工具。 该模块采用Grid-Threading Monte Carlo方法寻找最佳拟合结构，效率较高，与该领域其他软件相比，拟合时间减少了一两个数量级。 2.1.通过单粒子冷冻电子显微镜进行结构研究。 丙酮酸脱氢酶(PDH)2多酶复合物在细胞代谢中发挥核心作用,催化丙酮酸氧化脱羧为乙酰辅酶A,连接糖酵解和三羧酸循环。在这里，我们扩展了之前的研究，报告了含有 60 个 E2 和 E3 酶各 60 个拷贝的 1:1 化学计量复合物的结构。我们对纯化的 E2E3 复合物的重建三维结构进行了冷冻电子显微镜分析，并使用自动对接方法根据 E2 和 E3 分子的可能定位来解释密度图。我们的研究结果表明，丙酮酸脱氢酶复合物外壳中 E1 和 E3 分子的排列非常相似，并且表明环形间隙的设计允许硫辛酰结构域能够访问 E1、E2 和 E3 的活性位点来自环形间隙内的酶。 2.2 单粒子电子断层扫描结构测定 单粒子电子断层扫描可用于获取分子系统的 3D 体积数据，而无需对许多粒子进行平均。 该技术具有研究单个粒子的多种构象状态的潜力。 由于采样有限而导致的高噪声以及由于缺失楔形而导致的失真是该技术的两个困难。 在处理如此高的噪声数据时，普通的拟合方法常常会失败。 我们开发并在 CHARMM 中实现的核心加权拟合方法可以容忍如此高的噪声并产生合理的拟合结果。 我们正在应用核心加权拟合方法从单粒子电子断层扫描数据中推导出复杂的结构。 基于二十面体对称性的 PDH 复合物是最大的细胞机器之一，是单粒子电子断层扫描研究的良好候选者。 通过断层扫描图像，我们成功对接了E2CD核心和E1结构域，并获得了与cro-EM研究结果一致的复合物。 这些结果验证了实验技术以及我们对断层扫描数据的拟合方法。 另一个正在进行的项目是确定 HIV 包膜尖峰的结构。 HIV-1 的 Env 刺突由表面包膜糖蛋白 (gp120SU) 和跨膜包膜糖蛋白 (gp41TM) 亚基组成，其天然结构可引发部分有效的体液免疫反应，并代表病毒预防剂的明显疫苗靶标。 Env 尖峰被认为是三聚体，并且已经提出了基于结构的模型。然而，尽管付出了巨大的努力，原生 Env 尖峰内循环删除的 gp120 核心原子结构的排列和方向以及它们与 gp41 亚基的关联在很大程度上仍然是推测性的。最近，Subramaniams 博士的实验室获得了完整的、化学固定的 SIV 和 HIV-1 上的 Env 尖峰的电子断层图。我们现在正在应用图像处理和拟合方法来揭示单个病毒颗粒上 Env 尖峰的分布模式，并生成 SIV Env 尖峰的三维 (3D) 模型。 2.3 通过趋化受体组件的结构确定 我们实验室开发的图像处理方法已成功应用于通过电子断层扫描和电子断层扫描分段体积密度的平均来确定大肠杆菌细胞膜中趋化轴受体的三维结构。 这项工作弥合了全细胞晶体学和冷冻电子断层扫描之间的差距，并在理解细菌趋化性方面取得了重大进展。 尽管细菌化学感受器的周质和胞质部分的原子结构已被确定，但整个膜嵌入分子的整体结构仍未解决，高级寡聚复合物中的受体排列也是如此。 我们的结果证实了之前提出的受体的三聚体-二聚体排列。 此外，我们揭示了该信号转导机制的两种构象状态，为信号转导机制的研究提供了直接的结构信息。 3. 与Map对象进行蛋白质-蛋白质对接。 我们开发了一种使用图谱对象进行分子建模的方法，可以有效地从实验图谱中获取结构信息，并方便地操作图谱对象，直接使用图谱对象进行构象搜索。 这项开发工作已实施到 CHARMM 中。 该实现使CHARMM能够操作地图对象，包括地图输入、输出、比较、对接等。特别是，我们实现了核心加权相关函数以有效识别复杂地图中组件地图的正确拟合，以及网格线程蒙特卡罗从电子密度图有效构建复杂结构的搜索算法。 詹姆斯·M 博士Gruschus 正在将这种方法应用于过氧化氧还蛋白复合物的结构研究中。 过氧化还原蛋白 (Prx) 是几类蛋白质之一，可减少过氧化物（细胞代谢副产品产生的活性氧）。 为了对复合物进行建模，使用 EMAP 将 ATP 结合的人硫氧还蛋白 (hSrx) 与超氧化的 Prx II 对接。 在 Prx 半胱氨酸-亚磺酸残基最接近 hSrx 反应性半胱氨酸的对接结构中，Prx II 的 Asn186 与 hSrx 结合的 ATP β 和 γ 磷酸基团接触。 4. X射线晶体学 Parry 博士报道了 II 类主要组织相容性复合物 DRA、DRB3 与整联蛋白肽的复合物（PDB 代码 2Q6W）的结构。这是 DRB3 基因座的第一个结构。该结构以高分辨率模拟了与妊娠、新生儿、输血、干细胞和组织移植相关的严重血液疾病的分子基础。 第二种结构是具有改变的配体的复合物，已被精炼并等待提交。